Infectious bursal disease virus Vero cell-adapted strain and application thereof
A chicken infectivity, bursa of fabric technology, applied in the direction of antiviral agents, viruses/phages, viral antigen components, etc. High titer, protection of vaccinated animals, long-lasting effect
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Embodiment 1
[0026] Example 1 The acquisition and characteristic determination of IBDV virus
[0027] Suspected chicken infectious bursal disease material isolated from chicken flocks in Jiangsu, my country, was used to inoculate specific pathogen-free (Specific Pathogen Free, SPF) virus isolated from chicken embryos. Plaque purification was performed on chicken embryo fibroblasts, and the virus NJ strain was finally obtained through purification and screening of different plaques.
[0028] The NJ strain of the virus was identified for the following characteristics:
[0029] (1) Virus NJ strain amplification: Dilute the virus NJ strain with sterile PBS buffer for 10 3 After doubling, the 10-11-day-old SPF chicken embryos were inoculated by the chorioallantoic membrane route, the inoculation volume was 0.2 mL / embryo, cultured at 37°C, and the allantoic fluid of the chicken embryos was harvested within 36-48 hours.
[0030] (2) EID of the virus 50 : The chick embryo allantoic fluid obtaine...
Embodiment 2
[0034] Example 2 Screening of chicken infectious bursal virus Vero cell-adapted strain
[0035] In this implementation, chicken infectious bursal virus NJ strain was propagated on Vero cells cultured without serum, and a strain of IBDV virus NJ-23 with high virus titer and stable genetic characteristics was obtained through screening by limiting dilution method.
[0036] specific method:
[0037] (1) Vero cells were cultured with IVT medium (serum-free medium, purchased from Gibico) to a confluence of 95%, digested with 0.1% trypsin and centrifuged at 1000rpm, and resuspended in fresh IVT medium . Count the cells according to 8×10 5 96-well plate was laid in cell / well.
[0038] (2) Screening: Dilute NJ strain virus liquid with IVT medium added with whey protein hydrolyzate (Sigma) at a final concentration of 0.25-2.5% (mass percentage concentration), and use 5 dilutions (10 -4 ~10 -8 ) virus liquid infection step (1) spread the monolayer of Vero cells in a 96-well plate, ...
Embodiment 3
[0049] Example 3 Cultivate IBDV virus NJ-23 strain virus liquid on the Vero cells of serum-free microcarrier suspension culture
[0050] The method for cultivating the IBDV virus NJ-23 strain virus liquid on the Vero cells of serum-free microcarrier suspension culture is as follows:
[0051] (1) Serum-free culture of Vero cells: Culture Vero cells in IVT medium. When the cells reach 95% confluence, digest with 0.1% trypsin and centrifuge at 1000rpm, resuspend the cells with fresh IVT medium, and dissolve the cells Transfer to new culture flasks according to a certain ratio, continue to culture until the cells reach 95% confluence, and obtain Vero cells.
[0052] (2) Initial Vero cells were cultured in serum-free microcarrier suspension: the virus maintenance solution was obtained after adding whey protein hydrolyzate at a final concentration of 0.25% (mass percentage concentration) to the IVT medium, and its pH value was 7.1. The Vero cells obtained in step (1) were digested ...
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